Compositions and methods for treating lymphoma

ABSTRACT

This invention provides methods for treating neoplasias in a mammal. In particular, the invention provides methods for treating various types of lymphomas, including relapsed forms of non-Hodgkin&#39;s Lymphoma. These methods involve the administration of liposome-encapsulated vinca alkaloids, e.g., vincristine, to a mammal with a lymphoma.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application is a continuation of U.S. Ser. No. 07/541,436,now U.S. Pat. No. 6,723,338 which claims priority to U.S. ProvisionalPatent Application Nos. 60/127,444, filed Apr. 1, 1999, and 60/137,194,filed Jun. 2, 1999, each of which is incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for treatment of aneoplasia in a mammal, and in particular, relapsed forms of neoplasias.

BACKGROUND OF THE INVENTION

Despite years of research into the development of new methods oftreatment, cancers of the lymphatic system, or lymphomas, remain quitecommon. For example, more than 60,000 people in the United States arediagnosed with lymphoma each year, including more than 55,000 cases ofnon-Hodgkin's Lymphoma (NHL), and these numbers are constantlyincreasing. In addition, the prognosis for those affected by thesediseases is often poor, as the survival rates for lymphoma patientsremain low. Clearly, new methods for treating these diseases are needed.

While traditional treatments for lymphoma typically depend on the typeof lymphoma as well as the medical history of the patient, first-linetreatment for many lymphomas typically includes chemotherapy. Suchchemotherapy will often entail the administration of a “cocktail” ofcompounds, e.g., the formulation CHOP, which includes cyclophosphamide,doxorubicin, vincristine, and prednisone. In addition, certainfirst-line cancer treatments also include other forms of cancer therapy,such as radiation therapy.

In many cases, patients respond initially to such first-line treatments,but subsequently suffer a relapse, i.e., a tumor reappears or resumesgrowing. Following one such relapse, patients are often treated withfurther chemotherapy, e.g., with CHOP or with other formulations, or, insome cases, the patients are treated with other procedures such as bonemarrow transplantation. Again, in many cases, patients initially respondto such additional treatments, but subsequently suffer another relapse.In general, the more relapses a patient suffers, the less agreementthere is in the art concerning optimal subsequent treatment. In othercases, a patient fails to respond at all to a treatment, even initially,and is thus said to have a refractory cancer. In such cases as well,little agreement exists in the art regarding optimal subsequenttreatment.

Alkaloids isolated from the periwinkle plant (Vinca rosea), called“vinca alkaloids,” have proven effective for first line treatments ofmany types of lymphomas, leukemia, and other cancers. One such vincaalkaloid, vincristine, is included in the common chemotherapeuticformulation CHOP. Vincristine, which depolymerizes microtubules andthereby inhibits cell proliferation, is administered in its free form inCHOP. Liposome-encapsulated vincristine has been reported (see, e.g.,U.S. Pat. No. 5,741,516, or U.S. Pat. No. 5,714,163). In particular,these patents discuss the use of vincristine encapsulated inphosphatidylcholine, distearoylphosphatidylcholine, or sphingomyelin, inaddition to cholesterol. Successful clinical applications of thistechnology, however, have never been achieved. Indeed, major theoreticaland practical uncertainties remain, including uncertainties regardingbiodistribution, toxicity, and efficacy.

Lipid-encapsulated drug formulations may provide advantages overtraditional drug-delivery methods. For example, some lipid-basedformulations provide longer half-lives in vivo, superior tissuetargeting, and decreased toxicity. Numerous methods have been describedfor the formulation of lipid-based drug delivery vehicles (see, e.g.,U.S. Pat. No. 5,741,516). No studies, however, have demonstrated thatsuch liposome-encapsulated vinca alkaloid formulations offer anyadvantages over previous treatments, or have efficacy in the in vivotreatment of cancer in a patient. As such, there remains a need in theart for new methods for treating these diseases. Quite surprisingly, thepresent invention provides such methods.

SUMMARY OF THE INVENTION

It has now been discovered that liposome-encapsulated vinca alkaloids,such as vincristine, are especially efficacious in first line treatmentof neoplasia as well as for the treatment of relapsed forms ofneoplasias, in particular for lymphomas such as non-Hodgkin's Lymphomas.Provided herein, therefore, are methods for the treatment of these andother cancers.

In one aspect, this invention provides a method for treating a relapsedcancer in a mammal, the method comprising administering to the mammal apharmaceutical composition comprising a liposome-encapsulated vincaalkaloid. In one embodiment, the relapsed cancer is a non-Hodgkin'sLymphoma.

In another aspect, the present invention provides a method of treating anon-Hodgkin's Lymphoma in a patient, the method comprising administeringto the patient a pharmaceutical composition comprising aliposome-encapsulated vinca alkaloid, wherein the composition is free ofcardiolipin.

In one embodiment, the non-Hodgkin's Lymphoma is a member selected fromthe group consisting of aggressive NHL, transformed NHL, indolent NHL,relapsed NHL, refractory NHL, low grade non-Hodgkin's Lymphoma,follicular lymphoma, large cell lymphoma, B-cell lymphoma, T-celllymphoma, Mantle cell lymphoma, Burkitt's lymphoma, NK cell lymphoma,diffuse large B-cell lymphoma, and acute lymphoblastic lymphoma.

In one embodiment, the vinca alkaloid is vincristine, vinblastine,vinorelbine, or vindesine. In another embodiment, the liposome comprisesdistearoylphosphatidylcholine or sphingomyelin. In another embodiment,the liposome further comprises cholesterol. In another embodiment, theliposome comprise a pH gradient. In another embodiment, the pH at theinterior of the liposomes is lower than the pH at the exterior.

In another embodiment, the mammal is a human. In another embodiment, themammal has previously undergone at least one chemotherapy treatment. Inanother embodiment, the chemotherapy treatment comprised administrationof a free-form vinca alkaloid, such as vincristine, vinblastine,vindesine, or vinorelbine. In other embodiments, the chemotherapytreatment included an anthracycline-containing combination therapy. Inone such embodiment, the anthracycline was doxorubicin. In anotherembodiment, the mammal has exhibited a partial or complete response tothe chemotherapy prior to a relapse of the cancer. In anotherembodiment, the relapse is a second relapse.

In another embodiment, the liposome-encapsulated vinca alkaloid isadministered systemically by intravenous delivery. In anotherembodiment, the liposome-encapsulated vincristine is co-administeredwith cyclophosphamide, doxorubicin, and prednisone, forming CHOP (or, inthis case, “lipo-CHOP”). In another embodiment, theliposome-encapsulated vinca alkaloid is co-administered with at leastone additional anti-tumor agent. In another embodiment, the additionalanti-tumor agent is an anti-tumor monoclonal antibody, such as Oncolym™,Rituxan™, or Bexxar™. In another embodiment, the additional anti-tumoragent is an antisense drugs or an anti-tumor vaccine. In anotherembodiment, the liposome-encapsulated vinca alkaloid is co-administeredwith a prophylactic or therapeutic treatment for neurotoxicity, such asNeurontin™ gabapentin (Neurotonin).

In another embodiment, the liposome-encapsulated vinca alkaloid isadministered to the mammal once every 7-21 days, preferably every 14days. In another embodiment, the liposome encapsulated vinca alkaloid isadministered at a dosage falling within a range of about 1.4 to about2.4 mg/m².

The present invention provides an improvement on conventional methods oftreating cancer. In particular, the present invention provides a methodfor treating an aggressive, relapsed, transformed, indolent, orrefractory lymphoma in a mammal, the improvement comprisingadministering a liposome-encapsulated vinca alkaloid such as vincristine(or other liposome-encapsulated therapeutic agent) to the mammal. Inaddition, the present invention provides a basis for an improvedcombination chemotherapy for use in first-line treatment ofnon-Hodgkin's Lymphoma.

Kits including the herein-described formulations, and for preparing theherein-described formulations, as well as instructions for their use arealso included.

The present invention also provides the use of a liposome-encapsulatedvinca alkaloid in the preparation of a medicament for the treatment of aneoplasia, including non-Hodgkin's Lymphoma. In certain uses, theneoplasia is a relapsed, indolent, aggressive, or transformed neoplasia,e.g., non-Hodgkin's Lymphoma. In other uses, the medicament is used as afirst line treatment for a neoplasia. In preferred uses, the vincaalkaloid is vincristine. In other preferred uses, the vinca alkaloid ispresent in the medicament at a dosage, e.g., of about 2.4 to about 3.4mg/m², and is administered once every 7-21 days, most preferably every14 days.

Definitions

“Neoplasia,” as used herein, refers to any aberrant growth of cells,tumors, malignant effusions, warts, polyps, nonsolid tumors, cysts andother growths. A site of neoplasia can contain a variety of cell types,including but not limited, to neoplastic cells, vascular endothelia, orimmune system cells, such as macrophages and leukocytes, etc.

A “cancer” in a mammal refers to any of a number of conditions caused bythe abnormal, uncontrolled growth of cells. Cells capable of causingcancer, called “cancer cells”, possess a number of characteristicproperties such as uncontrolled proliferation, immortality, metastaticpotential, rapid growth and proliferation rate, and certain typicalmorphological features. Often, cancer cells will be in the form of atumor, but such cells may also exist alone within a mammal, or may be anon-tumorigenic cancer cell, such as a leukemia cell. A cancer can bedetected in any of a number of ways, including, but not limited to,detecting the presence of a tumor or tumors (e.g., by clinical orradiological means), examining cells within a tumor or from anotherbiological sample (e.g., from a tissue biopsy), measuring blood markersindicative of cancer (e.g., CA125, PAP, PSA, CEA, AFP, HCG, CA 19-9, CA15-3, CA 27-29, LDH, NSE, and others), and detecting a genotypeindicative of a cancer (e.g., TP53, ATM, etc.). However, a negativeresult in one or more of the above detection methods does notnecessarily indicate the absence of cancer, e.g., a patient who hasexhibited a complete response to a cancer treatment may still have acancer, as evidenced by a subsequent relapse.

“Systemic delivery,” as used herein, refers to delivery that leads to abroad bio-distribution of a compound within an organism. Systemicdelivery means that a useful, preferably therapeutic, amount of acompound is exposed to most parts of the body. To obtain broadbio-distribution generally requires a route of introduction such thatthe compound is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site. Systemic delivery of liposome-encapsulatedvinca alkaloids is preferably obtained by intravenous delivery.

“Lymphoma” refers to a malignant growth of B or T cells in the lymphaticsystem. “Lymphoma” includes numerous types of malignant growths,including Hodgkin's Lymphoma and non-Hodgkin's lymphoma (NHL).“Non-Hodgkin's Lymphoma” refers to a malignant growth of B or T cells inthe lymphatic system that is not a Hodgkin's Lymphoma (which ischaracterized, e.g., by the presence of Reed-Sternberg cells in thecancerous area). Non-Hodgkin's lymphomas encompass over 29 types oflymphoma, the distinctions between which are based on the type of cancercells. The particular classification depends on the particular system ofclassification used, such as the Working formulation, the Rappaportclassification, and the REAL classification. In preferred embodiments,the REAL classification is used.

A “relapsed cancer” or lymphoma refers to a cancer or lymphoma that hasrecurred following prior complete or partial remission in response to aprior treatment. Recurrence can be defined in any way, including areappearance or re-growth of a tumor as detected by clinical,radiological, or biochemical assays, or by an increased level of acancer marker. Prior treatments can include, but are not limited to,chemotherapy, radiation therapy, and bone marrow transplantation.

An “indolent” non-Hodgkin's Lymphoma is a classification that includesslow growing forms of lymphoma. They encompass what are called low gradeand some categories of intermediate grade NHL in the WorkingFormulation. Indolent NHLs are sometimes not responsive to conventionalcancer therapies such as chemotherapy and radiation therapy.

A “transformed” non-Hodgkin's Lymphoma is a classification sometimesemployed to describe an indolent NHL which acquires an aggressive aspectand becomes more responsive to standard chemotherapies.

Patients with “refractory cancer” or “refractory lymphoma” are those whohave failed to achieve complete remission on their first course ofcombination chemotherapy, or to patients who have failed to achievecomplete or partial remission on subsequent chemotherapy. “Primaryrefractory” patients are those who have never achieved completeremission even at first treatment.

A “stable disease” is a state wherein a therapy causes cessation ofgrowth or prevalence of a tumor or tumors as measured by the usualclinical, radiological and biochemical means, although there is noregression or decrease in the size or prevalence of the tumor or tumors,i.e., cancer that is not decreasing or increasing in extent or severity.

“Partial response” or “partial remission” refers to the amelioration ofa cancerous state, as measured by tumor size and/or cancer markerlevels, in response to a treatment. Typically, a “partial response”means that a tumor or tumor-indicating blood marker has decreased insize or level by about 50% in response to a treatment. The treatment canbe any treatment directed against cancer, but typically includeschemotherapy, radiation therapy, hormone therapy, surgery, cell or bonemarrow transplantation, immunotherapy, and others. The size of a tumorcan be detected by clinical or by radiological means. Tumor-indicatingmarkers can be detected by means well known to those of skill, e.g.,ELISA or other antibody-based tests.

A “complete response” or “complete remission” means that a cancerousstate, as measured by, for example, tumor size and/or cancer markerlevels, has disappeared following a treatment such as chemotherapy,radiation therapy, hormone therapy, surgery, cell or bone marrowtransplantation, or immunotherapy. The presence of a tumor can bedetected by clinical or by radiological means. Tumor-indicating markerscan be detected by means well known to those of skill, e.g., ELISA orother antibody-based tests. A “complete response” does not necessarilyindicate that the cancer has been cured, however, as a complete responsecan be followed by a relapse.

“Chemotherapy” refers to the administration of chemical agents thatinhibit the growth, proliferation and/or survival of cancer cells. Suchchemical agents are often directed to intracellular processes necessaryfor cell growth or division, and are thus particularly effective againstcancerous cells, which generally grow and divide rapidly. For example,vincristine depolymerizes microtubules, and thus inhibits cells fromentering mitosis. In general, chemotherapy can include any chemicalagent that inhibits, or is designed to inhibit, a cancerous cell or acell likely to become cancerous. Such agents are often administered, andare often most effective, in combination, e.g., in the formulation CHOP.

“Radiation therapy” refers to the administration of radioactivity to ananimal with cancer. Radiation kills or inhibits the growth of dividingcells, such as cancer cells.

“Surgery” is the direct removal or ablation of cells, e.g., cancercells, from an animal. Most often, the cancer cells will be in the formof a tumor (e.g., resulting from a lymphoma), which is removed from theanimal.

“Hormone therapy” refers to the administration of compounds thatcounteract or inhibit hormones, such as estrogen or androgen, that havea mitogenic effect on cells. Often, these hormones act to increase thecancerous properties of cancer cells in vivo.

“Immunotherapy” refers to methods of enhancing the ability of ananimal's immune system to destroy cancer cells within the animal.

A “free-form” therapeutic agent, or “free” therapeutic agent, refers toa therapeutic agent that is not liposome-encapsulated. Usually, a drugis presumed to be “free, or in a “free-form,” unless specifiedotherwise. A vinca alkaloid in free form may still be present incombination with other reagents, however, such as other chemotherapeuticcompounds, a pharmaceutical carrier, or complexing agents, i.e. as usedherein the term only specifically excludes lipid formulations of thevinca alkaloids.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides results for a clinical trial using the herein-describedmethods, in particular regarding the efficacy of the methods in thetreatment of indolent, transformed, relapsed, and aggressive postbone-marrow transplant (BMT) forms of non-Hodgkin's Lymphoma.

FIG. 2 provides results concerning the response to liposomal vincristinein Relapsed Aggressive NHL, particularly with regard to the effect ofthe prior regimen number.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

This invention provides methods of treating neoplasia in a patient. Thisinvention is based on the discovery that liposome-encapsulated vincaalkaloids are unusually effective in the treatment of a variety of formsof lymphoma. In particular, the surprising discovery was made that theadministration of liposome-encapsulated vinca alkaloids increases themedian survival of patients with lymphoma. In a particularly preferredembodiment, vincristine, encapsulated in a sphingomyelin and cholesterolbased liposome, is used in the treatment of non-Hodgkin's Lymphoma,especially relapsed forms of non-Hodgkin's Lymphoma (NHL). The inventionalso provides, inter alia, methods of treating indolent, transformed,and aggressive forms of NHL.

Often, such treatments of relapsed, indolent, transformed, andaggressive forms of non-Hodgkin's Lymphoma are administered following atleast one course of a primary anti-cancer treatment, such aschemotherapy and/or radiation therapy, followed by at least one partialor complete response to the at least one treatment. In otherembodiments, the liposomal vinca alkaloids are administered as a firstline treatment. In any of these embodiments, the liposome-encapsulatedvinca alkaloids can be provided as a single agent or in a combinationtherapy.

The present invention further provides dosages and dose scheduling ofliposomal vinca alkaloids for treatment of solid and non-solid tumorswith reduced toxicity.

I. Cancers Treatable with Lipid-Encapsulated Vinca Alkaloids

The methods described herein can be used to treat any type of cancer. Inparticular, these methods can be applied to cancers of the blood andlymphatic systems, including lymphomas, leukemia, and myelomas.

In preferred embodiments, the present methods are used to treat any ofthe large number of lymphomas. For example, both Hodgkin's andnon-Hodgkin's Lymphomas can be treated using the methods describedherein. In particularly preferred embodiments, the methods are used totreat non-Hodgkin's Lymphoma (NHL), including any type of NHL as definedaccording to any of the various classification systems such as theWorking formulation, the Rappaport classification and, preferably, theREAL classification. Such lymphomas include, but are not limited to,low-grade, intermediate-grade, and high-grade lymphomas, as well as bothB-cell and T-cell lymphomas. Included in these categories are thevarious types of small cell, large cell, cleaved cell, lymphocytic,follicular, diffuse, Burkitt's, Mantle cell, NK cell, CNS, AIDS-related,lymphoblastic, adult lymphoblastic, indolent, aggressive, transformedand other types of lymphomas. The methods of the present invention canbe used for adult or childhood forms of lymphoma, as well as lymphomasat any stage, e.g., stage I, II, III, or IV. The various types oflymphomas are well known to those of skill, and are described, e.g., bythe American Cancer Society (see, e.g., www3.cancer.org).

The methods described herein are also preferably applied to any form ofleukemia, including adult and childhood forms of the disease. Forexample, any acute, chronic, myelogenous, and lymphocytic form of thedisease can be treated using the methods of the present invention. Inpreferred embodiments, the methods are used to treat Acute LymphocyticLeukemia (ALL). More information about the various types of leukemia canbe found, inter alia, from the Leukemia Society of America (see, e.g.,www.leukemia.org).

Additional types of tumors can also be treated using the methodsdescribed herein, such as neuroblastomas, myelomas, prostate cancers,small cell lung cancer, and others.

II. First-Line Treatments

In numerous embodiments of the present invention, liposome-encapsulatedvinca alkaloids will be used as a first-line treatment for cancer. Inpreferred embodiments, liposome-encapsulated vinca alkaloids are used totreat lymphoma, particularly non-Hodgkin's Lymphoma. As used herein,“first-line treatment” refers to a primary treatment for a patientpresenting with a cancer, in contrast to a relapsed or refractorycancer.

In such embodiments, the liposome-encapsulated vinca alkaloids can beused alone or, preferably, in combination with other chemotherapeuticagents, such as cyclophosphamide, doxorubicin, and prednisone.Particularly preferred is the use of liposome encapsulated vincristinealong with cyclophosphamide, doxorubicin, and prednisone, therebyforming an improved, liposomal CHOP formulation (“lipo-CHOP.”)

When used as a single agent in first-line treatment, dosages and dosescheduling is preferably the same as single agent treatment for relapsedcancer. When used in combination regimes, dosages and dose schedulingmay be revised to correspond to the preferred regimen for thecombination.

III. Relapsed or Refractory Forms of the Diseases

The present methods can be used to treat primary, relapsed, transformed,or refractory forms of cancer. Often, patients with relapsed cancershave undergone one or more treatments including chemotherapy, radiationtherapy, bone marrow transplants, hormone therapy, surgery, and thelike. Of the patients who respond to such treatments, they may exhibitstable disease, a partial response (i.e., the tumor or a cancer markerlevel diminishes by at least 50%), or a complete response (i.e., thetumor as well as markers become undetectable). In either of thesescenarios, the cancer may subsequently reappear, signifying a relapse ofthe cancer.

In certain embodiments, the methods provided herein will be used totreat a patient that has undergone a single course of treatment for acancer, has partially or completely responded to such treatment, and hassubsequently suffered a relapse. In other embodiments, patients aretreated who have undergone more than one course of treatment, haveresponded more than once, and have subsequently suffered more than onerelapse. The previous course of treatment can include any anti-cancertreatment, including chemotherapy, radiation therapy, bone marrowtransplant, etc.

In certain embodiments of the present invention, liposomal alkaloids areemployed against “resistant” cancers, i.e., cancers which havepreviously exhibited a complete response to a treatment, but whichsubsequently manifest a resistance to second or later course oftreatment.

IV. Vinca and Other Alkaloids

The present invention can include the use of any naturally occurringalkaloid, including vinca alkaloids, or any synthetic derivative of anaturally occurring alkaloid. Vinca alkaloids include, but are notlimited to, vinblastine, vincristine, vindo line, vindesine, vinleurosine, vinrosidine, vinorelbine, or derivatives thereof (see, e.g., theMerck Index, 11^(th) Edition (1989) entries 9887, 9891, and 9893, forvinblastine, vincristine, and vindoline). Examples of other suitablealkaloids include, but are not limited to, the podophyllins,podophyllotoxins, and derivatives thereof (e.g., etoposide, etoposidephosphate, teniposide, etc.), the camptothecins (e.g., irinotecan,topotecan, etc.) the taxanes (taxol, etc.), and derivatives thereof. Allof the above compounds are well known to those of skill and are readilyavailable from commercial sources, by synthesis, or by purification fromnatural sources.

In preferred embodiments, the vinca alkaloid used in the presentinvention is vincristine. Vincristine, also known as leurocristinesulfate, 22-oxovincaleukoblastine, Kyocristine, vincosid, vincrex,oncovin, Vincasar PFS®, or VCR, is commercially available from any of anumber of sources, e.g., Pharmacia & Upjohn, Lilly, IGT, etc. It isoften supplied as vincristine sulfate, e.g., as a 1 mg/mL solution.

The present invention can comprise the use of a single vinca alkaloid ormultiple, co-administered vinca alkaloids. In addition, the one or morevinca alkaloids can be combined with other compounds or molecules, suchas other anti-neoplastic agents. In certain embodiments, suchcombinations of vinca alkaloids and/or other compounds can be made priorto liposomal formulation, thereby creating a combination within a singleliposome. In other embodiments, liposome-encapsulated vinca alkaloidsare formulated and subsequently combined with the other molecules, whichcan themselves be free-form or liposome-encapsulated.

Any of the therapeutic agents described herein, includingliposome-encapsulated alkaloids, can be subjected to pre-clinicaltesting in well known models of human diseases. In vivo models of humanlymphoma include mice carrying the non-Hodgkin's B-cell line DoHH2(Kluin-Nelemans H C, et al. (1991) Leukemia 5(3) 221-224), or micecarrying Daudi or Raji cell xenografts (see, for example Hudson, W A etal. (1998) Leukemia 12(12): 2029-2033). Many other oncological modelscan also be used and are known to those skilled in the art.

V. Lipids

Any of a number of lipids can be used to prepare the liposomes of thepresent invention, including amphipathic, neutral, cationic, and anioniclipids. Such lipids can be used alone or in combination, and can alsoinclude bilayer stabilizing components such as polyamide oligomers (see,e.g., U.S. Patent application “Polyamide Oligomers”, by Ansell, U.S.application Ser. No. 09/218,988, filed Dec. 22, 1998), peptides,proteins, detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S.application Ser. No. 08/485,608). In a preferred embodiment, cloakingagents, which reduce elimination of liposomes by the host immune system,can also be included, such as polyamide-oligomer conjugates, e.g.,ATTA-lipids, (see, U.S. patent application Ser. No. 08/996,783, filedFeb. 2, 1998) and PEG-lipid conjugates (see, U.S. patent applicationSer. Nos. 08/486,214, 08/316,407 and 08/485,608).

Any of a number of neutral lipids can be included, referring to any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH, includingdiacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

In preferred embodiments, the lipid used is sphingomyelin. Inparticularly preferred embodiments, the lipid comprises sphingomyelinand cholesterol. In such embodiments, the ratio of sphingomyelin tocholesterol is typically between about 75/25 (mol % sphingomyelin/mol %cholesterol) and about 50/50 (mol % sphingomyelin/mol % cholesterol),preferably between about 70/30 and 55/45 (mol % sphingomyelin/mol %cholesterol), and most preferably about 55/45 (mol % sphingomyelin/mol %cholesterol). Such ratios, may be altered, however, by the addition ofother lipids into the present formulations.

Cationic lipids, which carry a net positive charge at physiological pH,can readily be incorporated into liposomes for use in the presentinvention. Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);3β-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”); andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as LIPOFECTIN (including DOTMA andDOPE, available from GIBCO/BRL), LIPOFECTAMINE (comprising DOSPA andDOPE, available from GIBCO/BRL), and TRANSFECTAM (comprising DOGS, inethanol, from Promega Corp.).

Anionic lipids suitable for use in the present invention include, butare not limited to, phosphatidylglycerol, cardiolipin,diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, andother anionic modifying groups joined to neutral lipids.

In numerous embodiments, amphipathic lipids will be used. “Amphipathiclipids” refer to any suitable material, wherein the hydrophobic portionof the lipid material orients into a hydrophobic phase, while thehydrophilic portion orients toward the aqueous phase. Such compoundsinclude, but are not limited to, phospholipids, aminolipids, andsphingolipids. Representative phospholipids include sphingomyelin,phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatdylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, ordilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, suchas sphingolipids, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, can also be used. Additionally, such amphipathic lipidscan be readily mixed with other lipids, such as triglycerides andsterols.

The liposomes used in the present invention can be multilamellar orunilamellar, which can be formed using the methods disclosed herein andother methods known to those of skill in the art.

Also suitable for inclusion in the present invention are programmablefusion lipid formulations. Such formulations have little tendency tofuse with cell membranes and deliver their payload until a given signalevent occurs. This allows the lipid formulation to distribute moreevenly after injection into an organism or disease site before it startsfusing with cells. The signal event can be, for example, a change in pH,temperature, ionic environment, or time. In the latter case, a fusiondelaying or “cloaking” component, such as an ATTA-lipid conjugate or aPEG-lipid conjugate, can simply exchange out of the liposome membraneover time. By the time the formulation is suitably distributed in thebody, it has lost sufficient cloaking agent so as to be fusogenic. Withother signal events, its is desirable to choose a signal that isassociated with the disease site or target cell, such as increasedtemperature at a site of inflammation.

VI. Making Liposomes

A variety of methods are available for preparing liposomes as describedin, e.g., Szoka, et al., Ann. Rev. Biophys. Bioeng., 9:467 (1980), U.S.Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054,4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO91/17424, Deamer and Bangham, Biochim. Biophys. Acta, 443:629-634(1976); Fraley, et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979);Hope, et al., Biochim. Biophys. Acta, 812:55-65 (1985); Mayer, et al.,Biochim. Biophys. Acta, 858:161-168 (1986); Williams, et al., Proc.Natl. Acad. Sci., 85:242-246 (1988), the text Liposomes, Marc J. Ostro,ed., Marcel Dekker, Inc., New York, 1983, Chapter 1, and Hope, et al.,Chem. Phys. Lip., 40:89 (1986), all of which are incorporated herein byreference. Suitable methods include, but are not limited to, sonication,extrusion, high pressure/homogenization, microfluidization, detergentdialysis, calcium-induced fusion of small liposome vesicles, andether-infusion methods, all of which are well known in the art.

One method produces multilamellar vesicles of heterogeneous sizes. Inthis method, the vesicle-forming lipids are dissolved in a suitableorganic solvent or solvent system and dried under vacuum or an inert gasto form a thin lipid film. If desired, the film may be redissolved in asuitable solvent, such as tertiary butanol, and then lyophilized to forma more homogeneous lipid mixture which is in a more easily hydratedpowder-like form. This film is covered with an aqueous buffered solutionand allowed to hydrate, typically over a 15-60 minute period withagitation. The size distribution of the resulting multilamellar vesiclescan be shifted toward smaller sizes by hydrating the lipids under morevigorous agitation conditions or by adding solubilizing detergents, suchas deoxycholate.

Unilamellar vesicles can be prepared by sonication or extrusion.Sonication is generally performed with a tip sonifier, such as a Bransontip sonifier, in an ice bath. Typically, the suspension is subjected tosevered sonication cycles. Extrusion may be carried out by biomembraneextruders, such as the Lipex Biomembrane Extruder. Defined pore size inthe extrusion filters may generate unilamellar liposomal vesicles ofspecific sizes. The liposomes may also be formed by extrusion through anasymmetric ceramic filter, such as a Ceraflow Microfilter, commerciallyavailable from the Norton Company, Worcester Mass. Unilamellar vesiclescan also be made by dissolving phospholipids in ethanol and theninjecting the lipids into a buffer, causing the lipids to spontaneouslyform unilamellar vesicles. Also, phospholipids can be solubilized into adetergent, e.g., cholates, Triton X, or n-alkylglucosides. Following theaddition of the drug to the solubilized lipid-detergent micelles, thedetergent is removed by any of a number of possible methods includingdialysis, gel filtration, affinity chromatography, centrifugation, andultrafiltration.

Following liposome preparation, the liposomes which have not been sizedduring formation may be sized to achieve a desired size range andrelatively narrow distribution of liposome sizes. A size range of about0.2-0.4 microns allows the liposome suspension to be sterilized byfiltration through a conventional filter. The filter sterilizationmethod can be carried out on a high through-put basis if the liposomeshave been sized down to about 0.2-0.4 microns.

Several techniques are available for sizing liposomes to a desired size.One sizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles less than about 0.05 microns in size.Homogenization is another method that relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. The size of the liposomalvesicles may be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981),incorporated herein by reference. Average liposome diameter may bereduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Extrusion of liposome through a small-pore polycarbonate membrane or anasymmetric ceramic membrane is also an effective method for reducingliposome sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired liposome size distribution is achieved. Theliposomes may be extruded through successively smaller-pore membranes,to achieve gradual reduction in liposome size. For use in the presentinvention, liposomes having a size ranging from about 0.05 microns toabout 0.40 microns are preferred. In particularly preferred embodiments,liposomes are between about 0.05 and about 0.2 microns.

In preferred embodiments, empty liposomes are prepared usingconventional methods known to those of skill in the art.

Typically, as discussed infra, the liposomes used in the presentinvention will comprise a transmembrane potential, wherebyantineoplastic agents such as vinca alkaloids are effectively loadedinto and retained by the liposome. In preferred embodiments, thepotential will be effected by creating a pH gradient across themembrane. In particularly preferred embodiments, the pH is lower at theinterior of the liposomes than at the exterior. Such gradients can beachieved, e.g., by formulating the liposomes in the presence of a bufferwith a low pH, e.g., having a pH between about 2 and about 6, andsubsequently transferring the liposomes to a higher pH solution. Inpreferred embodiments, the pH is between about 3 and 5, and in mostpreferred embodiments, the pH is about 4. Any of a number of buffers canbe used, such as citrate.

Subsequently, before or after sizing, the external pH can be raised,e.g., to about 7 or 7.5, by the addition of a suitable buffer, such as asodium phosphate buffer. Raising the external pH creates a pH gradientacross the liposomal membrane, thereby promoting efficient drug loadingand retention.

Liposomes prepared according to these methods can be stored forsubstantial periods of time prior to drug loading and administration toa patient. For example, liposomes can be dehydrated, stored, andsubsequently rehydrated, loaded with one or more vinca alkaloids, andadministered. Dehydration can be accomplished, e.g., using standardfreeze-drying apparatus, i.e., they are dehydrated under low pressureconditions. Also, the liposomes can be frozen, e.g., in liquid nitrogen,prior to dehydration. Sugars can be added to the liposomal environment,e.g., to the buffer containing the liposomes, prior to dehydration,thereby promoting the integrity of the liposome during dehydration. See,e.g., U.S. Pat. No. 5,077,056 or 5,736,155.

In numerous embodiments, the empty liposomes are first formulated in lowpH buffer, and then manipulated in one of a variety of ways to obtainliposomes of the desired size. Methods for sizing liposomes includesonication, by bath or by probe, or homogenization. Preferably,following such treatments, the liposomes are between about 0.05 to 0.45microns. Most preferably, the liposomes are between about 0.05 and about0.2 microns. Such sized liposomes can then be sterilized by filtration.Also, particle size distribution can be monitored by conventionallaser-beam particle size discrimination or the like. In addition,methods of reducing liposome sizes to a relatively well defined sizedistribution are known, e.g., one or more cycles of extrusion of theliposomes through a small-pore polycarbonate membrane or an asymmetricceramic membrane.

VII. Preparation of Liposome-Encapsulated Vinca Alkaloids

Any of a number of methods can be used to load the vinca alkaloidsand/or other drugs into the liposomes. Such methods include, e.g., anencapsulation technique and a transmembrane potential loading method.Generally, following such methods, the vinca alkaloids are present atabout 0.1 mg/mL to about 0.5 mg/mL. Preferably, the vinca alkaloids arepresent at about 0.15 to 0.2 mg/mL.

In one encapsulation technique, the drug and liposome components aredissolved in an organic solvent in which all species are miscible andconcentrated to a dry film. A buffer is then added to the dried film andliposomes are formed having the drug incorporated into the vesiclewalls. Alternatively, the drug can be placed into a buffer and added toa dried film of only lipid components. In this manner, the drug willbecome encapsulated in the aqueous interior of the liposome. The bufferwhich is used in the formation of the liposomes can be any biologicallycompatible buffer solution of, for example, isotonic saline, phosphatebuffered saline, or other low ionic strength buffers. The resultingliposomes encompassing the vinca alkaloids can then be sized asdescribed above.

Transmembrane potential loading has been described in detail in U.S.Pat. Nos. 4,885,172; 5,059,421; 5,171,578; and 5,837,282 (which teachesionophore loading), each of which is incorporated herein by reference.Briefly, the transmembrane potential loading method can be used withessentially any conventional drug which can exist in a charged statewhen dissolved in an appropriate aqueous medium. Preferably, the drugwill be relatively lipophilic so that it will partition into theliposome membranes. A transmembrane potential is created across thebilayers of the liposomes or protein-liposome complexes and the drug isloaded into the liposome by means of the transmembrane potential. Thetransmembrane potential is generated by creating a concentrationgradient for one or more charged species (e.g., Na⁺, K⁺, and/or H⁺)across the membranes. This concentration gradient is generated byproducing liposomes having different internal and external media and hasan associated proton gradient. Drug accumulation can then occur in amanner predicted by the Henderson-Hasselbach equation.

Preferred methods of preparing liposome-encapsulated vinca alkaloids foruse in the present invention are discussed, e.g., in U.S. Pat. Nos.5,741,516, 5,814,335 and 5,543,152, each of which is assigned to InexPharmaceuticals Corp. and is incorporated herein by reference. In apreferred embodiment, liposomal vinca alkaloids are prepared prior touse from a kit including 3 or more vials. At least one of the vialscontains a vincristine solution containing, e.g., 1 mg/mL, 2 mg/mL, or 5mg/mL vincristine sulfate in buffer containing, e.g., 100 or 200 mg/mLmannitol (obtainable from, e.g., SP Pharmaceuticals LLC, Albuquerque,N.Mex.; other excipients that are pharmaceutically acceptable, and inwhich vincristine remains stable for extended periods, can also be used)and sodium acetate adjusted to pH 3.5 to 5.5, or preferably pH 4.5 to pH4.7. One of the vials contains a solution containing liposomescomprising sphingomyelin and cholesterol (each of which is commerciallyavailable, e.g., from NEN Life Sciences, Avanti Polar Lipids, etc.) andsuspended in a 300 mM citrate buffer at, e.g., pH 4.0. Another vial orvials contains a alkaline phosphate buffer (e.g., pH 9.0) such asdibasic sodium phosphate, 14.2 mg/ml (20 ml/vial).

In other preferred embodiments, a kit is used that contains 2 vialscontaining components that can be used to formulate the claimedliposome-encapsulated vincristine, or a kit containing 1 vial containinga stable preparation of liposomes comprising pre-loaded vincristine.Such stable preparations can be accomplished in any of a number of ways,including, but not limited to, (1) a hydrated preparation stored atambient temperatures or refrigerated and which contains one or moremodifications or components to enhance chemical stability, e.g.,antioxidants; (2) a hydrated preparation that was frozen and whichincludes a suitable excipient to protect from freeze/thaw-induceddamage; or (3) a lyophilized preparation. Typically, any of theabove-described kits also contain instructions for use as well asclean-up disposal materials.

To prepare the liposomes, the vincristine sulfate and liposome solutionsare each added to a sterile vial and mixed, at an appropriateconcentration ratio, e.g., 0.01/1.0 to 0.2/1.0 (wt. vinca alkaloid/wt.lipid). The mixture is mixed, e.g., by inverting the vial multipletimes. Following the formation of the liposomes in low pH buffer, andeither before or after the sizing of the liposomes, the liposomes areintroduced into buffer of a higher pH, e.g., a sodium phosphate buffer,thereby creating a pH gradient across the liposome surface. In preferredembodiments, the external environment of the liposomes is between aboutpH 7.0 and about pH 7.5. The liposomes and vinca alkaloids can be mixedfor an amount of time sufficient to achieve the desired alkaloid/lipidratio. The mixture can be mixed, e.g., by multiple inversions, andheated to temperatures between about 55° C. and about 80° C., preferablybetween about 60° C. and about 65° C., for about 5, 10, or more minutes.Such treatment causes greater than about 90% of the vincristine tobecome entrapped within the liposome.

In other embodiments, these steps are followed at a larger scale, andloaded liposomal vincristine is supplied to, e.g., a hospital pharmacyin ready-to-administer format. Such larger scale formulations may beprepared from different starting materials than those described for thekit; in particular, the buffers may be different.

VIII. Targeting Liposomes

In certain embodiments, it is desirable to target the liposomes of thisinvention using targeting moieties that are specific to a cell type ortissue. Targeting of liposomes using a variety of targeting moieties,such as ligands, cell surface receptors, glycoproteins, vitamins (e.g.,riboflavin) and monoclonal antibodies, has been previously described(see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044, the teachings ofwhich are incorporated herein by reference). The targeting moieties cancomprise the entire protein or fragments thereof.

Targeting mechanisms generally require that the targeting agents bepositioned on the surface of the liposome in such a manner that thetarget moiety is available for interaction with the target, for example,a cell surface receptor. The liposome is designed to incorporate aconnector portion into the membrane at the time of liposome formation.The connector portion must have a lipophilic portion that is firmlyembedded and anchored into the membrane. It must also have a hydrophilicportion that is chemically available on the aqueous surface of theliposome. The hydrophilic portion is selected so as to be chemicallysuitable with the targeting agent, such that the portion and agent forma stable chemical bond. Therefore, the connector portion usually extendsout from the liposomal surface and is configured to correctly positionthe targeting agent. In some cases, it is possible to attach the targetagent directly to the connector portion, but in many instances, it ismore suitable to use a third molecule to act as a “molecular bridge.”The bridge links the connector portion and the target agent off of thesurface of the liposome, thereby making the target agent freelyavailable for interaction with the cellular target.

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. patentapplication Ser. No. 08/316,394, filed Sep. 30, 1994, the teachings ofwhich are incorporated herein by reference. Examples of targetingmoieties can also include other proteins, specific to cellularcomponents, including antigens associated with neoplasms or tumors.Proteins used as targeting moieties can be attached to the liposomes viacovalent bonds (see, Heath, Covalent Attachment of Proteins toLiposomes, 149 Methods in Enzymology 111-119 (Academic Press, Inc.1987)). Other targeting methods include the biotin-avidin system.

IX. Administration of Lipid-Encapsulated Vinca Alkaloids

Liposome-encapsulated vinca alkaloids can be administered in any of anumber of ways, including parenteral, intravenous, systemic, local,intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation,or any such method of delivery. In preferred embodiments, thepharmaceutical compositions are administered intravenously by injection.In one embodiment, a patient is given an intravenous infusion of theliposome-encapsulated vinca alkaloids (single agent) through a runningintravenous line over, e.g., 30 minutes, 60 minutes, 90 minutes, orlonger. In preferred embodiments, a 60 minute infusion is used. Suchinfusions can be given periodically, e.g., once every 1, 3, 5, 7, 10,14, 21, or 28 days or longer, preferably once every 7-21 days, and mostpreferably once every 14 days. As used herein, each administration of aliposomal vinca alkaloid is considered one “course” of treatment.

Suitable formulation for use in the present invention can be found,e.g., in Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17^(th) Ed. (1985). Often, intravenous compositionswill comprise a solution of the liposomes suspended in an acceptablecarrier, such as an aqueous carrier. Any of a variety of aqueouscarriers can be used, e.g., water, buffered water, 0.4% saline, 0.9%isotonic saline, 0.3% glycine, 5% dextrose, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Often, normal buffered saline (135-150 mMNaCl) will be used. These compositions can be sterilized by conventionalsterilization techniques, such as filtration. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents, and the like, e.g.,sodium acetate, sodium lactate, sodium chloride, potassium chloride,calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.These compositions can be sterilized using the techniques referred toabove, or can be produced under sterile conditions. The concentration ofliposomes in the carrier can vary. Generally, the concentration will beabout 20-200 mg/mL, however persons of skill can vary the concentrationto optimize treatment with different liposome components or forparticular patients. For example, the concentration may be increased tolower the fluid load associated with treatment.

The amount of vinca alkaloids administered per dose is selected to beabove the minimal therapeutic dose but below a toxic dose. The choice ofamount per dose will depend on a number of factors, such as the medicalhistory of the patient, the use of other therapies, and the nature ofthe disease. In certain embodiments, an initially low dose will begiven, which can be increased based on the response and/or tolerance ofthe patient to the initial dose. For example, 0.5, 1.0, 1.5, 2.0, 2.4mg/m² (i.e., mg vinca alkaloid, e.g. ,vincristine, per m² body surfacearea) or higher concentrations can be administered. In preferredembodiments, patients are administered a dose of 2.0 mg/m²,corresponding to a lipid dose of about 40 mg/m² or about 1.1 mg/kg lipidand 0.05 mg/kg vincristine for an average 70 kg patient, or about 3 mgto about 6 mg vincristine per dose.

Patients typically will receive at least 2 courses of such treatment,and potentially more, depending on the response of the patient to thetreatment. In single agent regimens, total courses of treatment aredetermined by the patient and physician based on observed responses andtoxicity. Up to 12 courses of treatment, once every 14 days, havedemonstrated satisfactory patient responses. Greater numbers may bewarranted in certain cases. Similarly, the number of courses oftreatment using lipo-CHOP will be determined by the patient andphysician.

Because vincristine dosages are limited by neurotoxicity in humans, itis sometimes useful to co-administer liposomal vincristine with atreatment for neurotoxicity. This treatment may be prophylactic ortherapeutic. An example is the administration of Neurontin™ gabapentin(Parke-Davis), or neurotonin, for treatment of neuropathic pain, e.g.,100-200 mg Neurontin™ is administered 3 times per day to an adultpatient. If neuropathic pain improves, then liposomal vincristinetreatments may continue. Because this type of prophylactic ortherapeutic treatment is intended only to treat side-effects ofliposomal vincristine, it is considered separately from the combinationtherapies set forth below.

This invention is based in part on the surprising discovery that, incontrast to free form vinca alkaloids, liposome-encapsulated vincaalkaloids can be administered without a cap on the total dosage. Forexample, whereas free form vincristine is typically administered with acap of 2.0 mg, liposome-encapsulated vincristine can be administered ata constant dosage of, preferably, 2.0 mg/m². Thus, for a typical patientof from 1.5 to 3.0 m² surface area, a dose of from about 3.0 to about6.0 mg vincristine can be administered.

X. Combination Therapies

In numerous embodiments, liposome-encapsulated vinca alkaloids will beadministered in combination with one or more additional compounds ortherapies. For example, multiple vinca alkaloids can be co-administered,or one or more vinca alkaloids can be administered in conjunction withanother therapeutic compound, such as cyclophosphamide, doxorubicin,prednisone, other alkaloids such as the taxanes, camptothecins, and/orpodophyllins, other chemotherapeutic agents such as antisense drugs oranti-tumor vaccines. In a preferred embodiment, liposome-encapsulatedvincristine is co-administered with cyclophosphamide, doxorubicin, andprednisone. In certain embodiments, multiple compounds are loaded intothe same liposomes. In other embodiments, liposome-encapsulated vincaalkaloids are formed individually and subsequently combined with othercompounds for a single co-administration. Alternatively, certaintherapies are administered sequentially in a predetermined order, suchas in CHOP or lipo-CHOP. Liposome-encapsulated vincristine can also beformulated in a CVP combination, orcyclophosphamide-vincristine-prednisone.

Liposome-encapsulated vinca alkaloids can also be combined withanti-tumor agents such as monoclonal antibodies including, but notlimited to, Oncolym™ (Techniclone Corp. Tustin, Calif.) or Rituxan™(IDEC Pharmaceuticals), Bexxar™ (Coulter Pharmaceuticals, Palo Alto,Calif.), or IDEC-Y2B8 (IDEC Pharmaceuticals Corporation). In addition,liposome-encapsulated vinca alkaloids can be administered along with oneor more non-molecular treatments such as radiation therapy, bone marrowtransplantation, hormone therapy, surgery, etc.

In a preferred embodiment, liposome encapsulated vinca alkaloids areadministered in combination with an anti-cancer compound or therapywhich provides an increased or synergistic improvement in tumorreduction based on mechanism of action and non-overlapping toxicityprofiles. In particular, liposomal vinca alkaloids can be delivered witha taxane, which optionally may also be a liposomal taxane. While it isthought that vinca alkaloids depolymerize microtubules and taxanesstabilize microtubules, the two compounds have been found to actsynergistically in the impairment of tumor growth, presumably becauseboth are involved in the inhibition of microtubule dynamics. See,Dumontet, C. and Sikic, B. I. (1999) J. Clin Onc. 17(3) 1061-1070.Liposomal formulations of the vinca alkaloids according to the presentinvention will thus significantly diminish the myeloid and neurologictoxicity associated with the sequential administration of free formvinca alkaloids and taxanes.

Other combination therapies known to those of skill in the art can beused in conjunction with the methods of the present invention.

EXAMPLES

The following examples are offered to illustrate, but no to limit theclaimed invention.

Example 1 Making Liposome-Encapsulated Vincristine

Liposome-encapsulated vincristine (Vincristine Sulfate LiposomeInjection) was prepared using a six vial kit. Vials 1 and 2 contained avincristine sulfate solution (1 mg/mL Vincasar PFS®, SP PharmaceuticalsLLC, Albuqueque, N.Mex.) in buffer comprising mannitol and sodiumacetate, pH 4.5-4.7, vial 3 contained empty liposomes (100 mg/mLSphingomyelin/Cholesterol liposomes, at a ratio of between about 60/40to 50/50, or more preferably 55/45 mol %/mol %) in buffer comprising 300mM citrate at pH 4.0, vials 4 and 5 contained an alkaline phosphatebuffer (14.2 mg/mL dibasic sodium phosphate hepta hydrate), and vial 6was an empty, sterile vial. The foregoing empty liposomes were preparedusing thin film hydration and standard extrusion techniques, asdescribed in U.S. Pat. No. 5,741,516.

4 mL of Vincristine Sulfate was removed from vials 1 and 2 and added tosterile vial 6. Subsequently, 0.8 mL sphingomyelin/cholesterol liposomeswas removed from vial 3 and added to vial 6. Vial 6 was inverted fivetimes to mix the materials. 20 mL of the sodium phosphate solution fromvials 4 and 5 was added to vial 6. Vial 6 was again inverted five times,without shaking, to mix the materials. Vial 6 was then heated in a waterbath at 60-65° C. for five minutes, after which the vial was againinverted five times. The vial was then again heated for five minutes andinverted five more times.

The final product contained 0.16 mg/mL vincristine sulfate and 3.2 mg/mLtotal lipid.

Example 2 Liposome-Encapsulated Vincristine in Relapsed NHL Methods

50 patients with relapsed non-Hodgkin's Lymphoma (NHL), and 1 with AdultLymphoblastic Lymphoma (ALL), were included in the study. Each patientwas at least 16 years of age, did not have HIV or any other seriousinfection, did not have any disease of the central nervous system, andhad normal renal function and neutrophils at least 0.5K, and plateletsat least 50K. Each patient received up to 12 doses of 2.0 mg/m² ofintravenous liposomal-vincristine administered once every 14 days. Theliposomes used comprised sphingomyelin and cholesterol.

Results

35 of the 51 patients were evaluated. The median age of these 35patients was 62 years (range 19-86), and 21 of the patients were male.12 of the patients had follicular NHL, 7 had transformed, 11 diffuselarge cell, 3 mantle cell, 1 NK cell, and one ALL. Clinical grade washigh in 1, aggressive in 17, indolent in 10, and transformed in 7patients. Serum LDH was high in 16 out of the 35 patients, and B2microglobulin greater than 3.0 mg/L in 19 out of 30 patients. The mediannumber of prior therapeutic regimens was 3 (range 1-10). 18 of the 35patients were refractory to the regimen immediately preceding theliposome-encapsulated vincristine. All 35 had previously receivedvincristine administration. For the 34 patients with NHL, 14 patientsexhibited a complete or partial response, for an overall response rateof 40% (95% confidence interval: 24%-58%). Responses according toclinical grade was as shown in Table 1.

Transformed or Indolent Transformed Aggressive Aggressive # Patients 107 17 24 # Responders 1 5 8 13 (complete or partial response) % Completeor 10 71 47 54 partial response 95% Confidence 1–45 29–96 23–72 33–74Interval

Conclusions

Median response duration was 4 months. The fact that half of theresponding patients maintain the response for at least 4 months aftertreatment is a surprising, unexpected and clinically impressive responsefor a heterogeneous group of patients who previously would have beengiven a very poor prognosis.

The above results demonstrate that full doses of liposomal vincristinecan be given in relapsed NHL with good activity, even in heavilypretreated populations.

In addition, liposomal vincristine demonstrated significantly lessnon-specific toxicity than free vincristine. Peripheral neurotoxicity isthe most frequent and dose-limiting toxic effect of free vincristine.Peripheral neuropathic effects usually begin in adults who receive atotal dose of 5 to 6 mg (2-3 doses of free vincristine) and aregenerally significant after a cumulative dose of 15-20 mg (8-10 doses offree vincristine). Significantly, in the present study, a typicalpatient received 3-5 mg in one dose alone, and cumulative doses of up to37 mg were delivered, with no patient reporting significant liposomalvincristine-induced peripheral neurotoxicity. Even higher total dosesare likely to be tolerated. These higher doses are highly desirable forthe management of NHL, and represent a significant and surprising stepforward in the treatment of this disease.

Example 3 Use of Liposomal Vinca Alkaloids as First-line Treatment forLymphomas

This example illustrates the use of liposomal vinca alkaloids as afirst-line treatment, in combination with other chemotherapeutics, fortreatment of patients presenting with lymphomas, particularlynon-Hodgkin's lymphoma (low-grade or intermediate-grade). Patientspresenting with transformed or aggressive NHL may receive this improvedcombination treatment as a first-line treatment, or the physician mayprefer single agent OncoTCS™ treatment as described in the previousexamples. The combination therapy regimen set out below takes advantageof the surprising result that much higher doses of vincristine can beadministered when delivered in the liposomes of the present invention,with greatly reduced toxicity.

The preferred combination regimen is an improved CHOP regime(“Lipo-CHOP”) comprising: Cyclophosphamide, Hydroxydaunorubicin(doxorubicin), OncoTCS™, and Prednisone. One treatment cycle takes about5 days, and cycles are repeated about every 21-28 days. An exemplarcycle consists of

Cyclophosphamide (750 mg/m² IV, d 1)

Hydroxydaunorubicin (50 mg/m² IV, d 1)

OncoTCS™ (2.0 mg/m² IV, d 1, (no cap necessary))

Prednisone (100 mg PO qd×5 day)

Treatments are conducted with the same nursing interventions requiredfor standard CHOP treatment.

Patients receiving the improved CHOP treatment are expected to showsignificant improvement over standard CHOP in partial and completeremission rates, period of remission/time to relapse after treatment,and median survival times.

Example 4 Treatment of lymphomas with single agent liposomalvincristine.

In a further study, 50 human patients presenting with different classesof lymphoma were treated with single agent liposomal vincristine, asdescribed in Example 2. Results were as indicated in the followingchart:

First Multicenter Relapse Primary Post- ≧2 Study from CR Refractory ABMTRelapses Population* Number of 11 11 10 26 36 Patients Evaluable # CR 40 0 0 0 # PR 4 0 2 10 12 Overall 73 0 20 38 33 Response Rate (%) 95% 39to 95 0 to 28 1 to 32 20 to 59 Confidence Intervals (%) 18% grade 3 to 4neurotoxicity; no toxic deaths. CR = Complete response PR = Partialresponse Primary refractory means that no response to initial treatmentwas observed. ABMT = Autologous bone marrow transplant

Again, these results show that single agent treatment of liposomalvincristine is an excellent treatment for lymphomas. These resultsstrongly suggest a role for liposomal vincristine in Lipo-CHOP and forsingle agent first line treatment of lymphomas.

Example 5 Additional Studies

FIG. 1 provides results for a clinical trial using the herein-describedmethods, which demonstrates that the present methods are particularlyeffective in the treatment of indolent, transformed, relapsed, andaggressive post bone-marrow transplant (BMT) forms of non-Hodgkin'sLymphoma.

Example 6 Response to Liposomal Vincristine Per Prior Regimen Number

FIG. 2 provides results showing the number of evaluable patients withrelapsed aggressive NHL, the number of such patients that exhibited acomplete response or remission (CR), the number that exhibited a partialresponse or remission (PR), the percentage that exhibited either a CR ora PR, and the 95% confidence interval for each percentage value. Thesedata are presented for patients who have received one prior treatment,two or more prior treatments, and, of the latter category, those whoresponded to the treatment immediately prior to the study and those whodid not respond to the previous treatment.

This study demonstrates that the present methods are unusually effectivefor treating each category of patients.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. A kit for use in the treatment of a neoplasia in a mammal, said kitcomprising: (a) a first vial comprising a solution comprisingvincristine sulfate at a concentration of approximately 1 mg/ml,mannitol at a concentration of approximately 100 mg/ml, wherein saidsolution has a pH in the range of 3.5 to 5.5; (b) a second vialcomprising a citrate-buffered solution comprising liposomes comprisingsphingomyelin and cholesterol in a ratio of 75/25 (mol %sphingomyelin/mol % cholesterol) to 50/50 (mol % sphingomyelin/mol %cholesterol); (c) a third vial comprising a buffer solution comprisingdibasic sodium phosphate heptahydrate at a concentration ofapproximately 14.2 mg/ml; and (d) instructions for the use of said kitto prepare a liposomal composition useful in the treatment of aneoplasia in a mammal.
 2. A kit for use in the treatment of a neoplasiain a mammal, said kit comprising: (a) a first vial comprising avincristine solution, wherein said vincristine is present at aconcentration of 1 mg/ml to 5 mg/ml, wherein said solution has a pH inthe range of 3.5 to 5.5; (b) a second vial comprising liposomes in acitrate-buffered solution, wherein said liposomes comprise sphingomyelinand cholesterol in a ratio of 75/25 (mol % sphingomyelin/mol %cholesterol) to 50/50 (mol % sphingomyelin/mol % cholesterol), andwherein the pH of the interior and exterior of said liposomes is acidic;(c) a third vial comprising an alkaline phosphate buffer solution havinga pH higher than the pH of the solution of the second vial, such thatcombining the solutions of the second and third vials results in the pHof the exterior of said liposomes being neutral; and (d) instructionsfor the use of said kit to prepare liposomal compositions useful in thetreatment of a neoplasia in a mammal.
 3. A kit for use in the treatmentof a neoplasia in a mammal, said kit comprising: (a) a first vialcomprising a solution comprising vincristine at a concentration ofapproximately 1 mg/ml, mannitol at a concentration of approximately 100mg/ml, wherein said solution has a pH in the range of 3.5 to 5.5; (b) asecond vial comprising a citrate-buffered solution comprising liposomescomprising sphingomyelin and cholesterol in a ratio of 75/25 (mol %sphingomyelin/mol % cholesterol) to 50/50 (mol % sphingomyelin/mol %cholesterol); (c) a third vial comprising a buffer solution comprisingdibasic sodium phosphate heptahydrate at a concentration ofapproximately 14.2 mg/ml; and (d) instructions for the use of said kitto prepare a liposomal composition useful in the treatment of aneoplasia in a mammal.
 4. The kit of any one of claims 1-3, wherein saidneoplasia is a relapsed lymphoma or leukemia.
 5. The kit of any one ofclaims 1-3, wherein the pH of the interior and exterior of said liposomeis approximately 4.0.
 6. The kit of any one of claims 1-3, wherein saidliposome comprises sphingomyelin and cholesterol at a ratio of about55/45 (mol % sphingomyelin/mol % cholesterol).
 7. The kit of any one ofclaims 1-3, wherein the pH of the alkaline phosphate buffer solution isapproximately 9.0.